Mubmd 90031

The isolated cells were cultured in complete medium at 5000 cells/cm² within 6-well plates. When the cultured cells reached 95-100% confluence, three wells of cells were cultured with basal complete medium (as the control group), and the other three wells of cells were cultured with adipogenic differentiation medium (MUBMD-90031, Cyagen, CHN) (as the adipogenic group). The medium was changed every 3 days. After 7-day culture, qRT-PCR was performed for evaluating the expression of adipogenic genes (PPARγ, FABP4, LPL) in the cells. Meanwhile, the PPARγ expression was evaluated by immunofluorescence assay using an anti-PPARγ antibody (ab45036, Abcam, USA). The primer sequences were also listed in Table 3. Additionally, Oil red O was used to stain the neutral lipid vacuoles within cells for assessing the adipogenic differentiation of isolated cells after a 21-day culture. The steps for Oil red O staining assay are described below: the cells were washed twice with PBS and then fixed with 70% ethanol for 20 s. After that, they were incubated with filtered 0.3% Oil red O solution for 15 min, followed by thorough washing with PBS. The images of stained cells were viewed using a light microscope.

Adipogenic and osteogenic differentiation of ADSCs were performed as previously reported.7 For adipogenesis, cells were incubated in adipogenic medium (Cyagen, MUBMD‐90031) for 21 days. The medium was changed every three days. Adipogenesis was assessed by Oil Red O solution to stain lipids. For osteogenesis, cells were incubated in osteogenic medium (Cyagen, MUBMD‐90021) for 21 days. The medium was changed every three days. Osteogenesis was evaluated by alizarin red staining solution.

BMSCs (osteogenic induction: 1 × 10⁵ cells per well; adipogenic induction: 2 × 10⁵ cells per well) were seeded in 48-well plates and cultured for 24 h in complete medium, which was then replaced by osteogenic or adipogenic medium (MUBMD-90021 or MUBMD-90031; Cyagen Biosciences) supplemented with solvent, YB-EVs, AB-EVs, YB-OCY-EVs, AB-OCY-EVs, OC-CM, OC^YB-CM, OC^AB-CM, EVs from OC^YB-CM or OC^AB-CM, EVs-depleted OC^AB-CM, ALE (10 µM; 129318-43-0; Aladdin, Shanghai, China), OC^ALE-CM, OC^AB+ALE-CM, Y-Liver-EVs, A-Liver-EVs, Y-Ser-EVs, A-Ser-EVs, or AB-EVs pretreated with agomiR-483-5p, antagomiR-483-5p, agomiR-NC, or antagomiR-NC. The differentiation medium was changed every two days. For analyzing the expression of osteogenic or adipogenic genes, the cells were collected at 2 days after induction and processed for qRT-PCR. For detecting the formation of mineralized nodules or lipid droplets, the cells were stained with ARS solution (G1452; Solarbio) at 7 days after osteogenic induction or ORO solution (G1262; Solarbio) at 15 days after adipogenic induction. The percentages of ARS-positive (ARS⁺) and ORO⁺ areas were measured using Image-Pro Plus 6 software.